The present invention relates to a series of new phosphorous containing macrocyclic chelates which bind certain biological ions with relative specificity. The .sup.31 P resonance of these chelators shift to a new position in the NMR spectrum when a metal ion occupies the cavity of the chelate and the NMR chemical shift of the bound chelate differs for each metal ion. This property should be useful for monitoring the intracellular concentrations of certain biological cations including for example, Mg.sup.2+, Ca.sup.2+, and Zn.sup.2+, by .sup.31 P NMR. There are several fluorescent chelators commercially available for measuring intracellular cation concentrations and 1 or 2 fluorine containing chelators for NMR purposes. The fluorescent chelators will likely not be applicable for human studies and the .sup.19 F chelators are not as likely to be applied clinically as the .sup.31 P chelators of the present invention. Also, certain of the chelators when bound to Gd.sup.3+ has properties which should make it a safe, effective contrast agent for magnetic resonance imaging.
A great number of biological systems require the diamagnetic cations, Ca.sup.2+, Mg.sup.2+, and Zn.sup.2+ to regulate various reactions. The role of Ca.sup.2+ as an intracellular messenger ion in many types of cells is well established [1]. Mg.sup.2+ is a required cofactor for virtually all biological reactions involving ATP and may play an extensive role in the buffering of a greater variety of biological reactions [2]. Zn.sup.2+ is bound rather tightly in the active sites of several enzymes and its role appears to involve polarization of chemical bonds to aid bond hydrolysis, oxidation-reduction, or group transfer reactions. However, free Zn.sup.2+ may play a much wider role in some cells such as brain cells where it is known to be stored in neurons as storage granules and is mobilized during electrophysiologic activation [3].
An evaluation of the role of divalent cations in cell function has been limited by the availability of direct methods for measuring free cation concentration in cells and perfused organs. Presently available methods for measurement of divalent cations include indirect calculations based on equilibrium reactions [4], ion-selective micro-electrodes [5,6], and null point measurements using metallochromic dyes that are either microinjected into cells [7] or placed into the extracellular space with subsequent lysis of cells [8,9]. Virtually all of these methods are invasive in nature and require sample destruction prior to analysis. By contrast, NMR has made considerable advances as a non-invasive tool in measuring intracellular free divalent cation concentrations in perfused organs and intact cells.
Recently, fluorinated chelators have been effectively used to measure intracellular free Ca.sup.2+ [10,11] and Mg.sup.2+ [12,13] in cells and perfused organs by .sup.19 F NMR. These chelators work reasonably well in isolated cell systems but suffer from unexpected line broadening when used in perfused organs [13,14]. Another disadvantage with these systems is the synthetic routes to these compounds limits the possible chelate structures and hence metal-ion selectivity that may be designed into the chelate. Aspects of the present invention include the synthesis and development of a series of triaza and tetraaza macrocylic phosphonate monoesters and alkyl phosphinates as .sup.31 P NMR indicators to detect intracellular free cations in biological systems.
The present invention also relates to NMR imaging of living subjects, sometimes referred to as MRI (magnetic resonance imaging). More specifically, it relates to agents which can be used to enhance NMR contrast in such subjects.
Nuclear magnetic resonance (NMR) has been used for many years as a means of chemical analysis. NMR is a type of radio frequency spectroscopy which is based upon small energy differences between electrically charged atomic nuclei which are spinning parallel or antiparallel to an applied magnetic field. When radio frequency energy is applied to the sample, these spining atomic nuclei change spin states and in doing so, absorb some of the radio frequency energy. Nuclei in slightly different chemical environments within the same molecule change spin state at slightly different energies and this produces characteristic absorptions or resonances which help identify the molecular structure.
NMR has more recently been used in examinations of the human body. Other methods such as computerized axial tomography (CAT scanning) have been used in the past for this purpose, and still are. However, because NMR does not use ionizing radiation, it is believed to have some safety advantages over CAT. Thus, NMR is an advantageous method of producing cross-sectional images of the human body.
The quality of the images obtained from an NMR scan is based on two properties: the proton densities of the various tissues and differences in proton relaxation rates. The proton density of tissues cannot be readily altered. Proton relaxation rates can be adjusted by adding a paramagnetic relaxation agent, more commonly known as a "contrast agent." Contrast agents enhance the contrast in NMR images between magnetically similar but histologically dissimilar tissues.
Gadolinium has been tested as a contrast agent in the past because it has a large magnetic moment, which efficiently relaxes magnetic nuclei. Gadolinium's strong paramagnetic properties are the result of its seven unpaired electrons.
One drawback of gadolinium as a contrast agent is its toxicity to animals. One possible remedy for this problem is to incorporate gadolinium in a compound that would pass through the body and be excreted without releasing toxic gadolinium ions. Unfortunately, the rare earth elements, such as gadolinium, do not form stable covalent bonds with organic molecules, so such molecules can decompose in vivo and release the toxic ions.
There is a need for effective contrast agents which avoid the toxicity problems inherent in using gadolinium. Further, there is a need for new and better contrast agents, whether they include gadolinium or another paramagnetic metal.